Effects of Human Circulating Hematopoietic Stem & Progenitor Cells on Angiogenesis in the C32 Human Melanoma Xenograft Mouse Model Dillon Etter, Larry Solomon, George Sandusky, Karen Pollok, and Jamie Case Department of Pathology, Indiana University School of Medicine, Indianapolis Indiana ABSTRACT 7 Days Post-Injection Images 30 Days Post-Injection Images SUMMARY Tumor angiogenesis is a term used to describe the proliferation of blood vessels into a cancerous growth and is an essential process in the metastasis of a tumor. Recent studies suggest that endothelial cells involved in angiogenesis could be derived from bone marrow. Utilizing polychromatic flow cytometry (PFC), we isolated 2 distinct circulating hematopoietic stem and progenitor cell sub-populations. CD31+CD34brightCD45dimAC133+ cells termed pro-angiogenic circulating progenitor cells (PA-CPCs) and CD31+CD34brightCD45dimAC133- cells termed non-angiogenic CPCs (NA-CPCs). Previous findings show that injection of human PA-CPCs into immunodeficient mice with the C32 Human Melanoma Xenograft causes a significant increase in tumor growth, while injection of NA-CPCs does not. This experiment utilized immunohistochemistry techniques to investigate the vascular density at 7 days and 30 days in tumors of mice injected with PA-CPCs, NA-CPCs, and CD34+ cells. Rat anti-mouse CD31 antibody (Dianova) was used to stain mouse endothelial cells in the tumors and Image J software was utilized to quantify the results. At 7 days there was a significant increase in the vascular density, and hence the angiogenic potential of the tumors in mice injected with PA-CPCs compared to those injected with NA-CPCs, CD34+ cells or media. At 30 days, no significant difference in vascular denisty was observed among the four groups of mice. These data suggest that the increased growth of tumors in mice injected with PA-CPCs is due to increased angiogenesis in the early stages of tumor development. Anti-human and anti-mouse CD31, CD34, and CD33 antibodies were used to evaluate vasculature in the tumors. CD31 is an endothelial marker which stains both small and large vessels. CD33 stains myeloid progenitor cells, and CD34 stains adult hematopoietic stem cells. A previous experiment conducted at 28 days post-injection found that there was no human endothelium left in the xenograft after 28 days. Similarly, in this experiment, none of the xenografts harvested at 30 days displayed positive staining for human endothelium. Surprisingly, the xenografts harvested at 7 days were also negative for human cells as evidenced by a lack of anti-human CD31, CD34, and CD33 staining. This data suggests that although human PA- CPCs promote angiogenesis, they are no longer present in the tumor at 7 days. In our quantitative analysis of angiogenesis using a rat anti-mouse CD31 antibody (Dianova), increased staining was observed at 30 days compared to 7 days, suggesting an increase in the amount of murine vasculature between 7 and 30 days. Therefore, although human cells were no longer present at 7 days, the xenografts had not completely vascularized by 7 days. Additionally, out of the 7 day group, those animals that were injected with PA-CPCs exhibited a significant increase in murine vasculature compared to the NA-CPC, CD34+ and media groups. This confirms that PA-CPCs increase tumor angiogenesis. However, results from the 30-day study showed no notable difference in murine vasculature among the four groups of animals, proving that the effect of the human PA-CPCs is no longer visible at 30 days, and that tumors are fully vascularized by this time point. It has recently been reported that xenografts are 50% vascularized by 10 days. The data from this experiment supports that hypothesis. Data from a related experiment showns PA-CPCs also increase the weight and volume of the tumor (Figure 1 & 2). From our observations of xenograft tissues from mice receiving PA-CPCs and NA-CPCs, it was clear that tumors of mice receiving PA-CPCs exhibited less areas of necrosis than those of mice receiving NA-CPCs, CD34+ cells, and media (data not shown). This observation suggests that PA-CPCs could be used as a means of inhibiting necrosis in studies involving the use of xenograft transplants. Media CD34+ Non-Angiogenic CPC Pro-Angiogenic CPC Media CD34+ Non-Angiogenic CPC Pro-Angiogenic CPC BACKGROUND Mouse (+) Control Human (-) Control It has been reported that the ratio of PA-CPCs to NA-CPCs strongly correlates with the severity of the clinical state of patients with peripheral arterial disease (PAD). Patients with PAD exhibit a significant decrease in the ratio of PA-CPCs to NA-CPCs. Conversely, patients with tumor progression exhibit an increase in the ratio of PA-CPCs to NA-CPCs. These data suggest that CD31+CD34brightCD45dimAC133+ PA-CPCs are potential biomarkers for cardiovascular disease and tumor progression. The technique used to isolate these cells is known as PFC, which is used to sort rare cell sub-populations based on the surface markers or antigens they express. A novel PFC protocol has been developed to distinguish and isolate PA-CPCs and NA-CPCs based on AC133 expression. 30 Days Post-Injection Data Figure 1 Figure 2 MATERIALS and METHODS 7 Days Post-Injection Data Tissue specimens: NOD.CB17-Prkdcscid/J (NOD/SCID) mice were subcutaneously injected with 2x106 C32 human melanoma cells and tumor growth was monitored. Once tumors reached ~50mm3, mice were injected with 5x104 PA-CPCs, NA-CPCs, bulk CD34+ cells, or media/vehicle control (PBS). At 7 and 30 days post-injection, mice were euthanized and tumors harvested. Tissue preparation: Tissues were fixed overnight at room temperature in 10% neutral buffered zinc-formalin after which they were transferred through graded concentrations of alcohol to xylene. Tumors were then embedded in paraffin and cut into 6-µm sections. Sections were then mounted onto positively charged slides and baked at 60° C. Immunostaining: Tissues were deparaffinized and hydrated to distilled water. Antigen retrieval was achieved by boiling the slides for 10 minutes in the appropriate buffer (EDTA or citrate, depending on the antibody being used). Endogenous peroxidase activity was blocked using a 3% H2O2 solution. Tissues were incubated with a primary antibody followed by incubation with a biotinylated secondary antibody and avidin-biotin complex (Vector) or with a primary antibody followed by Biocare Mach III polymer kit. Visualization was achieved using DAB chromogen (Sigma). Tissues were counterstained in Mayer’s hematoxylin, dehydrated, and a cover slip was mounted using non-water soluble mounting media. Data Analysis: Images of stained slides at 2.5x magnification were analyzed using Image J software. For small tumors, one field was sufficient to capture a representative portion of the tumor. For large tumors, the average of multiple fields was calculated. Error bars represent the standard error, which was calculated by dividing the standard deviation of a group by the square root of the number of values in that group. CONCLUSIONS The mechanism by which PA-CPCs promote tumor growth is still not completely understood and warrants further investigation. Clarification of the function of these cells may permit their broader application to therapeutic and diagnostic procedures. The results of this study have shown that human PA-CPCs promote tumor angiogenesis and growth in mice harboring C32 Human Melanoma xenografts, and that these human cells are no longer present at 7 days. Furthermore, their pro-angiogenic effects, as indicated by differences in vascular density, are no longer visible by 30 days. Future studies should investigate angiogenesis at time points earlier than 7 days to determine whether the PA-CPCs promote angiogenesis by incorporation into the vasculature of the tumor, or by secretion of pro-angiogenic factors. Group Media CD34+ NA CPC PA CPC Amount of Positive CD31 Staining (Fraction of Low-Power Field) 0.016229098 0.003877163 0.013441358 0.009318775 0.009014428 0.023629583 0.019304854 0.01650121 0.02207757 0.020864198 0.015024599 0.007775422 0.012782735 0.016685258 0.008087355 0.005151721 0.00706326 0.014356354 0.014696736 0.005533563 0.007837648 0.030512875 0.017063065 0.022299383 0.014550365 0.01640872 0.011840578 Average 0.013433418 0.01626405 0.014042904 0.013806928 Standard Deviation 0.00598053 0.008737368 0.0040181 0.006886824 Standard Error 0.002674574 0.00390747 0.001640383 0.001910061 Group Media CD34+ NA CPC PA CPC Amount of Positive CD31 Staining (Fraction of Low-Power Field) 0.00126658 0.006898 0.007175 0.010894 0.00184788 0.003263 0.010159 0.018495 0.008421 0.004968 0.021472 0.003114 0.009379 0.017238 0.010562 0.005337 Mean 0.00155723 0.006452 0.007404 0.017025 Standard Deviation 0.00041104 0.003251 0.002332 0.004456 Standard Error 0.00029065 0.001454 0.001043 0.002228 REFERENCES Sanz, L., et al. 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